All vertebrates have multiple hemoglobins. During embryogenesis, there occurs one or more switches in the types of hemoglobin produced. Hereditary persistence of fetal hemoglobin is an example in humans in which the switching mechanism has failed. Hemoglobin switching involves not only the replacement of one hemoglobin type by another but also a concomitant change over in cell types. An unresolved issue is the extent to which the molecular and cellular switches are causally related. This proposal describes experiments designed to investigate the regulation of transcription in red blood cells during hemoglobin switching in the chicken, which takes place between 5 and 12 days of incubation. Red blood cells will be obtained from embryos at various stages during the switching period. Cell separation techniques will be used to fractionate the red blood cells according to both cell type (or lineage) and degree of cell maturity. The separated cells will then be analysed with respect to transcriptional specificity by means of isolating their nucleic and carrying out a transcriptional pulse-label in vitro. The highly radioactive RNA will then be assayed by hybridization to cloned DNA probes. The separated cells will also be assayed by hybridization for the content of specific globin sequences in nuclear and cytoplasmic RNA fractions. The principal objectives will be to test several predictions which have arisen from our preliminary studies. The main predictions are that (a) the first definitive cells (known for their synthesis of adult globins) to enter the circulation during embryo-genesis, are actively transcribing their embryonic globin genes but silencing the expression of these genes post-transcriptionally; (b) that these cells undergo an intrinsic switch to adult globin gene transcription during their maturation; (c) that the adult globin gene has two promoter sites for initiation of transcription which may be differentially regulated; and (d) that there is a developmentally regulated family of genes which are all related by being adjacent to members of a specific family of interspersed repetitive DNA. A new non-globin gene which we have found in the Beta-globin cluster will also be investigated.